Although Pichia pastoris is known for its ability to secrete high levels of recombinant proteins, actual yields vary widely for individual proteins. Consequently, GlycoFi's longterm objectives are focused on the improvement of protein yields in P. pastoris as part of the company's mission to develop yeast and fungi-based platforms for the production of therapeutic proteins with human glycosylation structures. The goal of this Phase I project is to develop and test a strategy for overcoming bottlenecks in the secretory process causing less than optimal yields, focusing specifically on improving translocation of the nascent peptide into the endoplasmic reticulum (ER) and protein folding within the ER. The project aims are to 1) assemble a library of fungal signal sequences, 2) create a library of human chaperone proteins, and 3) develop a screening method to test these signal sequences and chaperone proteins for increased secretion of a model protein. Taken together, Phase I aims will result in the creation of a toolkit for producing a more human-like secretory pathway in yeast strains expressing a recombinant protein. The toolkit will be utilized in Phase II in a high throughput screen to identify combinations of signal sequences and chaperones that optimize yield. Proposed Commercial Application: With a worldwide lack of manufacturing capacity blocking the development pipeline for new therapeutics, GlycoFi's technology will provide the biopharmaceutical industry with a high capacity alternative to mammalian cell culture that will allow life-improving drugs to reach the clinic in a faster, more cost-effective manner.